1. Field of the Invention
This invention relates generally to the field of biology and chemistry. More particularly, the invention is directed to fluorescent proteins.
2. Description of the Related Art
Fluorescent proteins including Green Fluorescent Protein (GFP), its mutants, and homologs are widely known today due to their intensive use as in vivo fluorescent markers in biomedical sciences as discussed in detail by Lippincott-Schwartz and Patterson in Science (2003) 300(5616):87-91.
Fluorescent proteins are proteins that exhibit fluorescence upon irradiation with light of the appropriate excitation wavelength. The fluorescent characteristic of these proteins is one that arises from the interaction of two or more amino acid residues of the protein, and not from a single amino acid residue.
The GFP from hydromedusa Aequorea aequorea (synonym A. victoria), described by Johnson et al. in J Cell Comp Physiol. (1962), 60:85-104, was found as a part of bioluminescent system of the jellyfish where GFP played role of a secondary emitter transforming blue light from photoprotein aequorin into green light. cDNA encoding A. victoria GFP was cloned by Prasher et al. (Gene (1992), 111(2):229-33). It turned out that this gene can be heterologously expressed in practically any organism due to unique ability of GFP to form a fluorophore by itself (Chalfie et al., Science 263 (1994), 802-805). This finding opens broad perspectives for use of GFP in cell biology as a genetically encoded fluorescent label.
The GFP was applied for wide range of applications including the study of gene expression and protein localization (Chalfie et al., Science 263 (1994), 802-805, and Heim et al. in Proc. Nat. Acad. Sci. (1994), 91: 12501-12504), as a tool for visualizing subcellular organelles in cells (Rizzuto et al., Curr. Biology (1995), 5: 635-642), for the visualization of protein transport along the secretory pathway (Kaether and Gerdes, FEBS Letters (1995), 369: 267-271).
A great deal of research is being performed to improve the properties of GFP and to produce GFP reagents useful and optimized for a variety of research purposes. New versions of GFP have been developed, such as a “humanized” GFP DNA, the protein product of which has increased synthesis in mammalian cells (Haas, et al., Current Biology (1996), 6: 315-324; Yang, et al., Nucleic Acids Research (1996), 24: 4592-4593). One such humanized protein is the “enhanced green fluorescent protein” (EGFP) mutant variant of GFP having two amino acid substitutions: F64L and S65T (Heim et al., Nature 373 (1995), 663-664). Other mutations to GFP have resulted in blue-, cyan- and yellow-green light emitting versions.
Despite the great utility of GFP, however, other fluorescent proteins with properties similar to or different from GFP would be useful in the art. In particular, benefits of novel fluorescent proteins include possibilities based on new spectra and better suitability for larger excitation. In 1999, GFP homologues were cloned from non-bioluminescent Anthozoa species (Matz et al., Nature Biotechnol. (1999), 17: 969-973). This discovery demonstrated that these proteins are not a necessary component of bioluminescence machinery. Anthozoa-derived GFP-like proteins showed great spectral diversity including cyan, green, yellow, red fluorescent proteins and purple-blue non-fluorescent chromoproteins (CPs) (Matz et al., Bioessays (2002), 24(10):953-959). Afterwards, cDNA of GFP-like proteins were cloned from several Hydroid jellyfishes and Copepods (Shagin et al., Mol Biol Evol. (2004), 21(5):841-850). GFP-like proteins already revealed over 120 fluorescent and colored GFP homologues. Similarity of these proteins to GFP ranges from 80-90% to less than 25% identity in amino-acid sequence.
The crystal structures of wild-type GFP and the GFP S65T mutant have been solved and reveal that the GFP tertiary structure resembles a barrel (Ormo et al., 1996, Science 273:1392-1395; Yang, et al., 1996, Nature Biotech 14: 1246-1251). The barrel consists of beta sheets in a compact anti-parallel structure, within which an alpha helix containing the chromophore is contained. The chromophore was confirmed to be formed by oxidative cyclization of three consecutive amino-acid residues, as was inferred from earlier biochemical studies (Cody et al., Biochemistry (1993) 32, 1212-1218). All GFP-like proteins tested share the same beta-can fold as GFP (Ormo et al. Science (1996) 273: 1392-1395; Wall et al. Nat Struct Biol (2000), 7: 1133-1138; Yarbrough et al. Proc Natl Acad Sci USA (2001) 98: 462-467; Prescott et al. Structure (Camb) (2003), 11: 275-284; Petersen et al. J Biol Chem (2003), 278: 44626-44631; Wilmann et al. J Biol Chem (2005), 280: 2401-2404; Remington et al. Biochemistry (2005), 44, 202-212; Quillin et al. Biochemistry (2005), 44: 5774-5787).
The utility of fluorescent proteins as a tool in molecular biology has prompted the search for other fluorescent proteins with different and improved properties, as compared to known fluorescent proteins. Thus, it is an object to provide novel fluorescent proteins that exhibit properties not currently available in the known fluorescent proteins, as well as DNAs encoding them.